Hepatitis C virus neutralizing antibody

ABSTRACT

A specific epitope on the surface of the hepatitis C virus that induces a neutralizing antibody response in vivo and neutralizing monoclonal antibodies that bind specifically to the epitope are disclosed. The antibodies block hepatitis C virus from infecting cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a 371 of PCT/US2013/041352 filed May 16,2013, which claims the benefit of U.S. Provisional Application Ser. No.61/648,386, filed May 17, 2012, under provisions of 35 U.S.C. 119 andthe International Convention for the Protection of Industrial Property,which are incorporated by reference in their entirety.

STATMENT OF GOVERNMENT SUPPORT

This invention was made in part with government support from theNational Institutes of Health. The government has certain rights in thisinvention.

BACKGROUND

Hepatitis C is an infectious disease affecting primarily the liver,caused by the hepatitis C virus (HCV). HCV is a major pathogentransmitted via infected blood that infects some 170 million peoplearound the world. The infection can remain hidden without showingsymptoms for years, and many people don't know they are infected.

A feature of HCV is that its course is unpredictable. The virus causeschronic (long-term) infections in 60% to 85% of infected individuals.From 20-50% of these infected individuals develop progressive liverdisease, leading ultimately to liver cirrhosis, liver failure and/orhepatocellular carcinoma. Liver damage from chronic hepatitis C virusinfection is now the most common cause of liver transplantation in theUS. However, a small minority of infected individuals seem to havesufficient immunity that they clear the virus soon after infection.

HCV is a positive-sense RNA virus belonging to the Flaviviridae family.It encodes a single polyprotein of ˜3,000 amino acids (aa). Through theaction of a combination of host and viral proteases, the polyprotein iscleaved into structural proteins (core, E1, E2, and p′7) andnonstructural proteins (NS2-NS5B). The two envelope glycoproteins, E1and E2, are believed to form heterodimers/oligomers on the surface ofHCV particles that participate in the process of cell entry (Bartosch,B. et al. 2003 J Exp Med 197:633-642).

HCV infection is treated with antiviral medications, e.g. pegylatedinterferon administered alone or in combination with ribavirin.Combination therapy with pegylated interferon and ribavirin is nowsuccessful in about half of the cases, but it is currently prohibitivelyexpensive, requires long-term treatment, and is associated with seriousside effects. In much of the world, such treatments are not economicallyfeasible. New direct-acting antiviral drugs such as protease andpolymerase inhibitors, either with or without interferon and/orribavirin, have the potential to increase the response rate and todecrease the duration of treatment. However, these drugs may also havesignificant side effects and are extremely expensive. Two proteaseinhibitors are now licensed for use in combination with interferon andribavirin although the treatment costs are between $26,000-$49,000 perpatient depending on the treatment duration, in addition to the costsfor pegylated interferon and ribavirin (Tungol, A. et al. J Manag CarePharm 2011; 17:685-94).

There are at least six known genotypes and more than 50 subtypes of HCV.Specific genotypes are in general located in distinct geographicallocations, while a small number of subtypes (1a, 1b, 2a and 3a) haverecently become more widely distributed and are associated with modernpractices such as medical injections, blood products and intravenousdrug use. Knowing the genotype can help predict the likelihood oftreatment response and, in many cases, determine the duration oftreatment. Patients with genotypes 2 and 3 are almost three times morelikely than patients with genotype 1 to respond to therapy with alphainterferon or the combination of alpha interferon and ribavirin. Whenusing combination therapy, the recommended duration of treatment dependson the genotype. For patients with genotypes 2 and 3, a 24-week courseof combination treatment is adequate, whereas for patients with genotype1, a 48-week course is recommended

Although a vaccine that prevents and treats HCV infection is urgentlyrequired, no vaccine is currently available for HCV. A therapeuticvaccine would be an invaluable adjunct to current treatment options forHCV.

One of the major challenges facing the development of treatments or avaccine for HCV is the high degree of genetic diversity that isexhibited by the virus, estimated to be 10 fold higher than that seen inHIV. Other factors that have hindered vaccine development for HCVinclude the lack of an accessible animal model and the fact that thevirus cannot be easily grown in the laboratory. Although it may not bepossible to develop a vaccine that targets all HCV genotypes, genotypespecific vaccines that are administered in regions where specificgenotypes dominate may be a realistic goal. Both T cell and antibodybased vaccines to prevent and also to treat HCV infection are underdevelopment.

Further, a major challenge facing HCV infected patients that undergoliver transplants is recurrence of hepatitis C virus infection followingotherwise technically successful liver transplantation. Recurrent HCVinfection leads to diminished graft and patient survival. Although anumber of predictors of severe recurrence have been identified, nodefinitive strategy has been developed to prevent recurrence. Althoughhepatitis B virus (HBV)-specific specific antibody products exist thatare effective in preventing recurrence of HBV infection in livertransplant patients, no HCV-specific antibody is available yet forpreventing recurrence of HCV infection in liver transplant patients.Currently, the only effective treatments for prevention of HCVrecurrence after liver transplantation are interferon-based therapies,administered alone or in combination with ribavirin.

There remains a need in the art for more treatments of and vaccines toprevent HCV infection.

SUMMARY

Disclosed herein is an HCV neutralizing antibody binding specifically toHCV E2 protein Epitope II (EPII).

In an embodiment, the antibody or fragment thereof comprises a heavychain variable region comprising at least one heavy chaincomplementarity determining region (CDR) amino acid sequence selectedfrom the group consisting of CDR1 comprising residues 25-32 (GYSFTNYY)of SEQ ID NO:2, CDR2 comprising residues 50-57 (IFPGGGNT) of SEQ IDNO:2, and CDR3 comprising residues 96-107 (SRDIY GDAWFAY) of SEQ IDNO:2. In an embodiment, the antibody or fragment thereof comprises alight chain variable region comprising at least one light chain CDRamino acid sequence selected from the group consisting of CDR1comprising residues 27-37 (QNIVHRNGNTY) of SEQ ID NO:3, CDR2 comprisingresidues 55-57 (KVS) of SEQ ID NO:3, and CDR3 comprising residues 94-102(FQGSHFPPT) of SEQ ID NO:3.

In an embodiment, the antibody or fragment thereof comprises a heavychain variable region comprising a heavy chain third complementaritydetermining region (CDR3) amino acid sequence comprising residues 96-107(SRDIYGDAWFAY) of SEQ ID NO:2. In an embodiment, the antibody orfragment thereof comprises a light chain variable region comprising alight chain CDR3 amino acid sequence comprising residues 94-102(FQGSHFPPT) of SEQ ID NO:3.

In an embodiment, the antibody or fragment thereof binds specifically tohepatitis C virus (HCV) E2 protein Epitope II (EP II), and comprises aheavy chain variable region comprising an amino acid sequence consistingof SEQ ID NO:2 or a light chain variable region comprising an amino acidsequence consisting of SEQ ID NO:3.

In an embodiment, the isolated antibody or a fragment thereof bindingspecifically to hepatitis C virus (HCV) E2 protein Epitope II (EP II)comprises a heavy chain encoded by a polynucleotide consisting of SEQ IDNO: 4; and a light chain encoded by a polynucleotide consisting of SEQID NO: 5.

Compositions comprising the antibody or fragment thereof and methods ofmaking and using the antibody or fragment thereof are also disclosed.

In an embodiment, a method of detecting HCV E2 protein Epitope IIcomprises contacting the antibody or fragment thereof with a sampleunder conditions such that the antibody binds an HCV E2 protein EpitopeII (EP II) sequence comprising at least residues 434-446 of SEQ ID NO:1;and detecting antibody bound to EP II.

In an embodiment, a method of preventing HCV infection comprisescontacting the antibody or fragment thereof to a cell that will beexposed to or infected with HCV.

In an embodiment, a method of treating or preventing HCV infectioncomprises administering the antibody or fragment thereof to a to asubject exposed to or infected with HCV

Hybridomas, polynucleotides encoding the antibody or fragment thereof,recombinant vectors, and host cells expressing the antibody or fragmentthereof are also disclosed.

These and other advantages of the invention, as well as additionalinventive features, will be apparent from the description of theinvention provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents peptide sequences and histograms showingpeptide-specificity of the monoclonal antibodies. Panel (A) presents theamino acid sequences of peptides used in the study. The sequence ofPeptide A corresponds to amino acid residues 412-447 of the HCVpolyprotein within the region of the E2 protein of HCV H strain (H77,genotype 1a) (residues 412-447 of SEQ ID NO:1) and was used to immunizemice to generate the monoclonal antibodies tested in this study. Thesequences of truncated forms of Peptide A, i.e., Peptide B, B short andPeptide D are also shown (the indicated residue numbers for eachsequence identify the relative position of the sequence in SEQ ID NO:1).The locations of Epitope I and Epitope II within Peptide A are alsoshown. Panel (B) is a histogram showing Peptide A-specificity of themonoclonal antibodies in an ELISA. The y axis indicates absorbance at405 nm obtained in the ELISA, representing specific binding of a givenantibody to Peptide A. Panel (C) is a histogram showing PeptideB-specificity of the monoclonal antibodies in an ELISA.

FIG. 2 presents histograms showing neutralization of chimeric viruses bymonoclonal antibodies in Huh 7.5 cells. Panel (A) is a histogram showingneutralization of genotype 1a/2a virus in which the x axis indicates theparticular antibody tested in the experiment and the y axis indicatesthe relative infectivity of the virus (%), i.e., percent of the negativecontrol (cell culture medium). Panel (B) presents histograms showingPeptide B-specific neutralization of genotype 1a/2a virus by antibody#41. Antibody #41 was adsorbed with (+) or without (−) Peptide B priorto performing an ELISA to test its binding to Peptide B (left panel),and a neutralization assay to assess its neutralizing activity in Huh7.5 cells (right panel). Each of these samples shown on the x axis wastested at the dilution of 1:10⁵ in an ELISA. The y axis indicates theabsorbance at 405 nm obtained in an ELISA, representing the specificbinding of a given antibody to Peptide B. The data shown represent atleast 3 independent experiments, with the error bars indicating thestandard error of the mean. For the neutralization assay (right panel),the supernatant was diluted at 1:400, and incubated with the genotype1a/2a virus before adding the mixture to Huh 7.5 cells. The cell culturemedium (Med) was used as the negative control against the testedantibodies. The x axis indicates the samples tested in this assay. The yaxis indicates the relative infectivity of the virus (%), i.e., percentof the negative control. The statistical significance of the differencein infectivity is also indicated. Panel (C) is a histogram showing theinability of the antibodies to cross-neutralize the J6/JFH1 virus, agenotype 2a virus.

FIG. 3 presents a histogram showing the inability of non-neutralizingantibodies (#12 and #50) to block virus neutralization by antibody #41.Results from three independent experiments are shown with the error barindicating the standard error of the mean.

FIG. 4 summarizes results of epitope mapping by screening random peptidephage-display libraries with the two neutralizing Peptide B-bindingantibodies. The candidate core residues at the epitope-paratope contactinterfaces are indicated in bold font. The symbol (x) denotes the aminoacid residue other than L at the position. The peptide sequences, fromtop to bottom, are SEQ ID NOs:6-13, respectively.

FIG. 5 presents identification of residues involved in antibodyrecognition by mutational analysis. Panel (A) indicates the mutatedsequences chemically synthesized and tested by ELISA. The sequence ofPeptide B short, is amino acids 434-446 of SEQ ID NO:1 and, as shownbelow the sequence, the B short mutant peptides contained a singlealanine (A) substitution at positions 437, 438, 440, 441 and 442,respectively. A hyphen indicates an amino acid residue in the mutantpeptides identical to that of the H77 sequence. Panel (B) is a histogramshowing detection of antibody #41 binding by ELISA with thebiotin-conjugated B short peptide and its mutants at 1:10⁵ dilution, andapplied as the primary antibody. PBS was included as the negativecontrol. The x axis indicates the mutation used in each assay. The yaxis indicates the absorbance at 405 nm, representing specific bindingof the antibody to each individual peptide. Data shown represent 3independent experiments with standard deviation indicated as error bars.

FIG. 6A presents a summary of the E2 protein amino acid residuesinvolved in binding of each antibody disclosed herein. FIG. 6B shows thesequence of the HCV 1a (H77) genotype from residues 412-447 of SEQ IDNO:1 and summarizes the alignment of amino acid sequences of the E2region 412-447 of various HCV genotypes below the sequence. Residuesidentified as involved in binding of the four antibodies disclosedherein are shown in the H77 sequence in bold and underlined letters. Inthe alignments, a hyphen indicates an amino acid residue identical tothat of the H77 sequence.

FIG. 7 shows the effect of the W437F switch on antibody binding. Panel(A) shows a schematic representation of the mutations of peptide B usedin the ELISA. Biotin-conjugated peptides were chemically synthesized torepresent Peptide B (residues 427-446 of SEQ ID NO:1), the truncatedPeptide B (B short) (residues 434-446 of SEQ ID NO:1), and B shortsequences with the indicated specific single mutations, F or A, atposition 437. A hyphen indicates an amino acid residue identical to thatof the H77 sequence. Panel (B) shows a histogram of results from theELISA. The x axis indicates the antibodies used in this assay. The yaxis indicates the absorbance obtained at 450 nm, which represents themeasurement of specific binding of a given antibody to each individualpeptide.

FIG. 8 shows the nucleic acid sequence (SEQ ID NO: 5) and translatedprotein sequence (SEQ ID NO: 3) of the kappa chain of antibody #41 andthe nucleic acid sequence (SEQ ID NO: 4) and translated protein sequence(SEQ ID NO: 2) of the heavy chain of antibody #41.

DETAILED DESCRIPTION

Novel monoclonal antibodies that bind to HCV E2 protein Epitope II (HCVEPII) and inhibit HCV infection of cells are provided herein. Inparticular, the antibodies described herein are capable of neutralizingHCV genotype 1a infectivity in a cell culture system. Therefore, theantibodies of the present invention are useful for treating orpreventing HCV infection.

In an embodiment, the isolated neutralizing antibody disclosed hereinspecifically binds HCV EPII. The antibody can be a monoclonal antibody.The antibody can be an intact antibody or an antigen-binding fragment ofthe antibody. The antibody can be a mouse, a goat, a sheep, a guineapig, a rat, or a rabbit antibody. In some embodiments, the antibody canbe a rodent antibody, specifically a mouse antibody. The antibody can bea chimeric antibody, specifically a humanized antibody. The HCV EPIIepitope recognized can be the HCV 1a genotype EPII, comprising W⁴³⁷. Insome embodiments, the HCV 1a genotype EPII recognized comprises W⁴³⁷ andL⁴³⁸

In an embodiment, the neutralizing antibody comprises a heavy chainvariable region amino acid sequence comprising SEQ ID NO:2. In anembodiment, the heavy chain variable region amino acid sequence is SEQID NO:2.

In an embodiment, the neutralizing antibody comprises a light (kappa)chain variable region amino acid sequence comprising SEQ ID NO:3. In anembodiment, the light chain variable region amino acid sequence is SEQID NO:3.

In an embodiment, the neutralizing antibody comprises a heavy chainvariable region encoded by SEQ ID NO:4. In an embodiment, theneutralizing antibody comprises a light (kappa) chain variable regionencoded by SEQ ID NO:5.

The isolated neutralizing antibody or antigen-binding fragment thereofcan comprise a heavy chain variable region comprising at least one heavychain complementarity determining region (CDR) amino acid sequenceselected from the group consisting of residues 25-32 (GYSFTNYY) of SEQID NO:2, 50-57 (IFPGGGNT) of SEQ ID NO:2, and 96-107 (SRDIY GDAWFAY) ofSEQ ID NO:2. In an embodiment, the heavy chain variable region comprisesCDR amino acid sequences consisting of residues 25-32 (GYSFTNYY) of SEQID NO:2, 50-57 (IFPGGGNT) of SEQ ID NO:2, and 96-107 (SRDIY GDAWFAY) ofSEQ ID NO:2.

The isolated neutralizing antibody or antigen-binding fragment thereofcan comprise a heavy chain variable region comprising a light chainvariable region comprising at least one light chain CDR amino acidsequence selected from the group consisting of residues 27-37(QNIVHRNGNTY) of SEQ ID NO:3, CDR2 comprising residues 55-57 (KVS) ofSEQ ID NO:3, and CDR3 comprising residues 94-102 (FQGSHFPPT) of SEQ IDNO:3. In an embodiment, the light chain variable region comprises CDRamino acid sequences consisting of residues 27-37 (QNIVHRNGNTY) of SEQID NO:3, CDR2 comprising residues 55-57 (KVS) of SEQ ID NO:3, and CDR3comprising residues 94-102 (FQGSHFPPT) of SEQ ID NO:3.

The term “neutralizing antibody” is an antibody that is capable ofkeeping an infectious agent, usually a virus, e.g., HCV, from infectinga cell by neutralizing or inhibiting its biological effect, for exampleby blocking the receptors on the cell or the virus. Neutralization canhappen when antibodies bind to specific viral antigens, blocking thepathogen from entering their host cells.

The term “antibody” or “immunoglobulin,” as used interchangeably herein,includes whole antibodies and any antigen binding fragment(antigen-binding portion) or single chain cognates thereof. An“antibody” comprises at least one heavy (H) chain and one light (L)chain. In naturally occurring IgGs, for example, these heavy and lightchains are inter-connected by disulfide bonds and there are two pairedheavy and light chains, these two also inter-connected by disulfidebonds. Each heavy chain is comprised of a heavy chain variable region(abbreviated herein as V_(H)) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(abbreviated herein as V_(L)) and a light chain constant region. Thelight chain constant region is comprised of one domain, CL. The V_(H)and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDR),interspersed with regions that are more conserved, termed frameworkregions (FR) or Joining (J) regions (JH or JL in heavy and light chainsrespectively). Each V_(H) and V_(L) is composed of three CDRs three FRsand a J domain, arranged from amino-terminus to carboxy-terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, J. The variableregions of the heavy and light chains bind with an antigen. The constantregions of the antibodies may mediate the binding of the immunoglobulinto host tissues or factors, including various cells of the immune system(e.g., effector cells) or humoral factors such as the first component(Clq) of the classical complement system.

The term “antigen-binding portion” or “antigen-binding fragment” of anantibody, as used herein, refers to one or more fragments of an antibodythat retain the ability to specifically bind to an antigen (e.g., HVC E2protein EPII). It has been shown that fragments of a full-lengthantibody can perform the antigen-binding function of an antibody.Examples of binding fragments denoted as an antigen-binding portion orfragment of an antibody include (i) a Fab fragment, a monovalentfragment consisting of the V_(L), V_(H), CL and CH1 domains; (ii) aF(ab')₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the V_(H) and CH1 domains; (iv) a Fv fragment consistingof the V_(L) and V_(H) domains of a single arm of an antibody, (v) a dAbincluding VH and VL domains; (vi) a dAb fragment (Ward et al. (1989)Nature 341, 544-546), which consists of a V_(H) domain; (vii) a dAbwhich consists of a VH or a VL domain; and (viii) an isolatedcomplementarity determining region (CDR) or (ix) a combination of two ormore isolated CDRs which may optionally be joined by a synthetic linker.Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions arepaired to form monovalent molecules (such a single chain cognate of animmunoglobulin fragment is known as a single chain Fv (scFv). Suchsingle chain antibodies are also intended to be encompassed within theterm “antibody fragment” . Antibody fragments are obtained usingconventional techniques known to those with skill in the art, and thefragments are screened for utility in the same general manner as areintact antibodies. Antigen-binding fragments can be produced byrecombinant DNA techniques, or by enzymatic or chemical cleavage ofintact immunoglobulins.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. In some embodiments, the term “monoclonal antibody”refers to an antibody derived from a single cell clone. Antigen bindingfragments (including scFvs) of such immunoglobulins are also encompassedby the term “monoclonal antibody” as used herein. Monoclonal antibodiesare highly specific, being directed against a single antigenic site.Furthermore, in contrast to conventional (polyclonal) antibodypreparations, which typically include different antibodies, directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single epitope on the antigen. Monoclonal antibodiescan be prepared using any art recognized technique and those describedherein such as, for example, a hybridoma method, a transgenic animal,recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), or usingphage antibody libraries using the techniques described in, for example,US Patent No. 7,388,088 and US patent application Ser. No. 09/856,907(PCT Int. Pub. No. WO 00/31246). Monoclonal antibodies include chimericantibodies, human antibodies and humanized antibodies and may occurnaturally or be produced recombinantly.The term “recombinant antibody,”refers to antibodies that are prepared, expressed, created or isolatedby recombinant means, such as (a) antibodies isolated from an animal(e.g., a mouse) that is transgenic or transchromosomal forimmunoglobulin genes (e.g., human immunoglobulin genes) or a hybridomaprepared therefrom, (b) antibodies isolated from a host cell transformedto express the antibody, e.g., from a transfectoma, (c) antibodiesisolated from a recombinant, combinatorial antibody library (e.g.,containing human antibody sequences) using phage display, and (d)antibodies prepared, expressed, created or isolated by any other meansthat involve splicing of immunoglobulin gene sequences (e.g., humanimmunoglobulin genes) to other DNA sequences. Such recombinantantibodies may have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies can be subjected to in vitro mutagenesisand thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

The term “chimeric immunoglobulin” or antibody refers to animmunoglobulin or antibody whose variable regions derive from a firstspecies and whose constant regions derive from a second species.Chimeric immunoglobulins or antibodies can be constructed, for exampleby genetic engineering, from immunoglobulin gene segments belonging todifferent species.

The term “human antibody,” as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from human germline immunoglobulin sequences asdescribed, for example, by Kabat et al. (See Kabat, et al. (1991)Sequences of proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).Furthermore, if the antibody contains a constant region, the constantregion also is derived from human germline immunoglobulin sequences. Thehuman antibodies may include amino acid residues not encoded by humangermline immunoglobulin sequences (e.g., mutations introduced by randomor site-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody”, as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences.

The human antibody can have at least one or more amino acids replacedwith an amino acid residue, e.g., an activity enhancing amino acidresidue that is not encoded by the human germline immunoglobulinsequence. Typically, the human antibody can have up to twenty positionsreplaced with amino acid residues that are not part of the humangermline immunoglobulin sequence. In a particular embodiment, thesereplacements are within the CDR regions as described in detail below.

The term “humanized immunoglobulin” or “humanized antibody” refers to animmunoglobulin or antibody that includes at least one humanizedimmunoglobulin or antibody chain (i.e., at least one humanized light orheavy chain). The term “humanized immunoglobulin chain” or “humanizedantibody chain” (i.e., a “humanized immunoglobulin light chain” or“humanized immunoglobulin heavy chain”) refers to an immunoglobulin orantibody chain (i.e., a light or heavy chain, respectively) having avariable region that includes a variable framework region substantiallyfrom a human immunoglobulin or antibody and complementarity determiningregions (CDRs) (e.g., at least one CDR, two CDRs, or three CDRs)substantially from a non-human immunoglobulin or antibody, and furtherincludes constant regions (e.g., one constant region or portion thereof,in the case of a light chain, and preferably three constant regions inthe case of a heavy chain). The term “humanized variable region” (e.g.,“humanized light chain variable region” or “humanized heavy chainvariable region”) refers to a variable region that includes a variableframework region substantially from a human immunoglobulin or antibodyand complementarity determining regions (CDRs) substantially from anon-human immunoglobulin or antibody.

A non-human antibody is humanized using a method known in the art. Ingeneral, a humanized antibody has at least one amino acid residueintroduced from a non-human donor. The humanization of a non-humanantibody may be performed by replacing CDR sequences of a human antibodywith corresponding CDR sequences of a non-human species, e.g., a rodentsuch as a mouse, having the desired specificity and affinity. Thus, ahumanized antibody is a chimeric antibody, and a region that is smallerthan the variable region of a substantially intact human antibody may bereplaced by the corresponding sequences from a non-human antibody. Forexample, a humanized antibody may be a human antibody in which some CDRresidues and possibly some framework (FR) residues are replaced byresidues from the analogous CDR and FR sites in antibodies of a rodent.

An “antigen” is an entity (e.g., a proteinaceous entity or peptide) towhich an antibody binds. In various embodiments disclosed herein, anantigen is a peptide derived from the HCV E2 protein comprising EpitopeII (“HCV EPII”) (a.a. 427-446 of the HCV polyprotein). For example, theantigen can be HCV E2 protein or a peptide comprising aa 427-446 of SEQID NO:1, such as Peptide A (FIG. 1A). In some embodiments, an antigen isHCV EPII Peptide B, or Peptide “B short” (FIG. 1A).

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody specifically binds.Epitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein. Epitopes formedfrom contiguous amino acids are typically retained on exposure todenaturing solvents, whereas epitopes formed by tertiary folding aretypically lost on treatment with denaturing solvents. An epitopetypically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or15 amino acids, often contiguous amino acids, in a unique spatialconformation. An epitope herein is not limited to a polypeptide havingthe exact sequence of the portion of the parent protein from which it isderived. Thus the term “epitope” encompasses sequences identical to thenative sequence, as well as modifications to the native sequence, suchas deletions, additions and substitutions (generally conservative innature) to which the antibody dislosed herein specifically binds.Methods for determining what epitopes are bound by a given antibody(i.e., epitope mapping) are well known in the art and include, forexample, immunoblotting and immunoprecipitation assays, whereinoverlapping or contiguous peptides from the antigen are tested forreactivity with the given antibody. The neutralizing monoclonalantibodies disclosed herein bind specifically to HCV EPII.

Methods of determining spatial conformation of epitopes are also wellknown in the art and include, for example, x-ray crystallography and 2-or more dimensional nuclear magnetic resonance.

The terms “specific binding,” “specifically binds,” “selective binding,”and “selectively binds” mean that an antibody exhibits appreciableaffinity for a particular antigen or epitope and, generally, does notexhibit significant cross-reactivity with other antigens and epitopes.“Appreciable” binding affinity includes binding with an affinity of atleast 10⁶ M⁻¹, specifically at least 10⁷ M⁻¹, more specifically at least10⁸ M⁻¹, yet more specifically at least 10⁹ M⁻¹, or even yet morespecifically at least 10¹° M⁻¹. A binding affinity can also be indicatedas a range of affinities, for example, 10⁶ M⁻¹ to 10¹° M⁻¹, specifically10⁷ M⁻¹ to 10¹⁰ M⁻¹, more specifically 10⁸ M⁻¹ to 10¹⁰ M⁻¹. An antibodythat “does not exhibit significant crossreactivity” is one that will notappreciably bind to an undesirable entity (e.g., an undesirableproteinaceous entity). An antibody specific for a particular epitopewill, for example, not significantly crossreact with other epitopes onthe same protein or peptide. Specific binding can be determinedaccording to any art-recognized means for determining such binding. Insome embodiments, specific binding is determined according to Scatchardanalysis and/or competitive binding assays.

The term “linked” used herein refers to a linkage of two entities, forexample a labeling material and an antibody, by covalent or non-covalentbonding. A linkage mediated by a linker molecule or the like is alsoincluded.

The term “toxic material” used herein refers to a material which can belinked to an antibody or a fragment thereof and can exert toxic effectson a target, such as a cancer cell. For example, radioactive materialssuch as yttrium-90, iodine-131, etc. and cytotoxic materials such ascalicheamicin are included among toxic materials.

The term “labeling material” used herein refers to a material whichbinds to an antibody or a fragment thereof and is detectable by aphysical or chemical method to permit identification of the location orquantity of the antibody or the fragment thereof. The labeling materialis used to label the antibody to make detection of bound or unboundantibody easy. Suitable detectable materials include a variety ofenzymes, prosthetic groups, fluorescent materials, light-emittingmaterials and radioactive materials. Examples of suitable enzymesinclude horseradish peroxidase, alkaline phosphatase, β-galactosidase oracetylcholinesterase. Examples of suitable prosthetic groups includestreptavidin/biotin and avidin/biotin. Examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin. Examples of light-emitting materials includeluminol, and examples of radioactive materials include 1251, 1311, 35S,and 3H. Detection of the labeling material can be performed by anyappropriate method known in the art.

The term “isolated” refers to a nucleic acid, a polypeptide, or othercomponent that is removed from components with which it is naturallyassociated. The term “isolated” can refer to a polypeptide that isseparate and discrete from the whole organism with which the molecule isfound in nature or is present in the substantial absence of otherbiological macro-molecules of the same type. The term “isolated” withrespect to a polynucleotide can refer to a nucleic acid molecule devoid,in whole or part, of sequences normally associated with it in nature; ora sequence, as it exists in nature, but having heterologous sequences inassociation therewith; or a molecule disassociated from the chromosome.

The term “isolated nucleic acid molecule” or “isolated polynucleotide”as used herein in reference to nucleic acids encoding antibodies orantibody fragments (e.g., V_(H), V_(L), CDR3), is intended to refer to anucleic acid molecule in which the nucleotide sequences are free ofother genomic nucleotide sequences, e.g., those encoding antibodies thatbind antigens other than HCV E2 protein EPII, which other sequences maynaturally flank the nucleic acid in human genomic DNA.

The term “nucleic acid molecule” or “polynucleotide” as used herein, isintended to include DNA molecules and RNA molecules. A nucleic acidmolecule may be single-stranded or double-stranded. A polynucleotide canbe obtained by a suitable method known in the art, including isolationfrom natural sources, chemical synthesis, or enzymatic synthesis.

An isolated polynucleotide encoding an antibody heavy chain variableregion having the amino acid sequence of SEQ ID NO: 2 is disclosed. Thepolynucleotide can comprise SEQ ID NO: 4.

An isolated polynucleotide encoding an antibody light chain variableregion having the amino acid sequence of SEQ ID NO: 3. Thepolynucleotide can comprise SEQ ID NO: 5.

The term “vector” used herein refers to a nucleic acid sequence toexpress a target gene in a host cell. Examples include a plasmid vector,a cosmid vector, a bacteriophage vector, and a viral vector. Examples ofviral vectors include a bacteriophage vector, an adenovirus vector, aretrovirus vector, and an adeno-associated virus vector.

For example, the vector may be an expression vector including a membranetargeting or secretion signaling sequence or a leader sequence, inaddition to an expression control element such as promoter, operator,initiation codon, termination codon, polyadenylation signal, andenhancer. The vector may be manufactured in various ways known in theart depending on the purpose. An expression vector may include aselection marker for selecting a host cell containing the vector.Further, a replicable expression vector may include an origin ofreplication.

The term “recombinant vector” used herein refers to a vector operablylinked to a heterologous nucleotide sequence for the purpose ofexpression, production and isolation of the heterologous nucleotidesequence. The heterologous nucleotide sequence can be a nucleotidesequence encoding all or part of the heavy chain or the light chain ofan antibody disclosed herein.

The recombinant vector may be constructed for use in prokaryotic oreukaryotic host cells. For example, when a prokaryotic cell is used as ahost cell, the expression vector used generally includes a strongpromoter capable of initiating transcription (for example, p_(L) ^(λ)promoter, trp promoter, lac promoter, tac promoter, T7 promoter), aribosome binding site for initiating translation, and atranscription/translation termination sequence. When a eukaryotic cellis used as a host cell, the vector used generally includes the origin ofreplication acting in the eukaryotic cell, for example f1 origin ofreplication, SV40 origin of replication, pMB1 origin of replication,adeno origin of replication, AAV origin of replication, or BBV origin ofreplication, but is not limited thereto. A promoter in an expressionvector for a eukaryotic host cell may be a promoter derived from thegenomes of mammalian cells (for example, a metallothionein promoter) ora promoter derived from mammalian viruses (for example, an adenoviruslate promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, acytomegalovirus promoter, and a tk promoter of HSV). A transcriptiontermination sequence in an expression vector for a eukaryotic host cellmay be, in general, a polyadenylation sequence.

The term “operably linked” refers to a nucleic acid sequence placed intoa functional relationship with another nucleic acid sequence. Forexample, DNA for a presequence or secretory leader is operably linked toDNA for a polypeptide if it is expressed as a preprotein thatparticipates in the secretion of the polypeptide; a promoter or enhanceris operably linked to a coding sequence if it affects the transcriptionof the sequence; or a ribosome binding site is operably linked to acoding sequence if it is positioned so as to facilitate translation.Generally, “operably linked” means that the DNA sequences being linkedare contiguous, and, in the case of a secretory leader, contiguous andin reading phase. However, enhancers do not have to be contiguous.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist, the synthetic oligonucleotide adaptors orlinkers are used in accordance with conventional practice. A nucleicacid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

A single vector can be used to simultaneously express both the heavychain and the light chain of the antibody. Alternatively, the heavychain and the light chain of the antibody can be expressed from twodifferent vectors. In the latter case, the two vectors may be introducedinto a single host cell by simultaneous transduction or targetedtransduction.

The host cell of the vector may be any cell that can be practicallyutilized by the expression vector. For example, the host cell may be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell. Further, the host cell may be a prokaryoticcell, such as a bacterial cell. A prokaryotic host cell may be aBacillus genus bacterium, such as E. coli JM109, E. coli BL21, E. coliRR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillussubtilis, and Bacillus thuringiensis; or an intestinal bacterium, suchas Salmonella typhimurium, Serratia marcescens, and various Pseudomonasspecies. A eukaryotic host cell may be a yeast (e.g., Saccharomycescerevisiae), an insect ell, a plant cell, or an animal cell, forexample, mouse Sp2/0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6,W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, or a MDCK cell line.

The polynucleotide or recombinant vector including the polynucleotidemay be transferred into the host cell using a method known in the art.For example, when a prokaryotic cell is used as the host cell, thetransfer may be performed using a CaCl₂ method or an electroporationmethod, and when a eukaryotic cell is used as the host cell, thetransfer may be performed by microinjection, calcium phosphateprecipitation, electroporation, liposome-mediated transfection, or genebombardment, but is not limited thereto.

Disclosed herein is a recombinant vector comprising a polynucleotideconsisting of SEQ ID NO: 4. Also disclosed is a recombinant vectorcomprising a polynucleotide consisting of SEQ ID NO: 5. A suitable hostcell can be transformed with one or both of the recombinant vectors orone or both of the polynucleotides.

A method of isolating the antibody from the host cell is also disclosed.In an embodiment the method comprises culturing the host cell andisolating from the culture an antibody binding to HCV EPII. The methodcan further comprise screening the antibody in a cell culture system todetermine that it is a neutralizing antibody. A genotype 1a HCV or achimeric HCV including gentoype 1a EPII can be used in the screeningassay to determine if the isolated antibody reduces infectivity of theHCV.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

As used herein, the term “subject” includes any human or non-humananimal. For example, the methods and compositions of the presentinvention can be used to treat a subject having cancer. In a particularembodiment, the subject is a human. The term “non-human animal” includesall vertebrates, e.g., mammals and non-mammals, such as non-humanprimates, sheep, dog, cow, chickens, amphibians, reptiles, etc.

The terms “treat” , “treating” and “treatment” mean implementation oftherapy with the intention of reduction in severity or frequency ofsymptoms, elimination of symptoms or their underlying cause, preventionof the occurrence of symptoms or their underlying cause, or improvementor remediation of damage.

The term “sample” refers to tissue, body fluid, or a cell from a patientor a subject. Normally, the tissue or cell will be removed from thesubject, but in vivo diagnosis is also contemplated.

The term “E2 polypeptide” is intended to refer to a molecule derivedfrom an HCV E2 region. The mature E2 region of HCV-Ia begins atapproximately amino acid 384, numbered relative to the full-length HCV-Ipolyprotein (SEQ ID NO:1). A signal peptide begins at approximatelyamino acid 364 of the polyprotein. The corresponding region for otherHCV genotypes and subtypes are known and readily determined bycomparison to the HCV-Ia polyprotein. For ease of discussion then,numbering herein is with reference to the HCV-Ia polyprotein, but it isto be understood that an “E2 polypeptide” also encompasses E2polypeptides from any of the various HCV genotypes, such as HCV-I,HCV-2, HCV-3, HCV-4, HCV-5 and HCV-6 and subtypes thereof, such asHCV-Ia, HCV-2a, HCV-3a, HCV-4a, HCV-5a and HCV-6a. Thus, for example,the term “E2” polypeptide refers to native E2 sequences from any of thevarious HCV genotypes, unless specifically identified, as well asanalogs, muteins and immunogenic fragments, as discussed further below.The complete genotypes of many of these strains are known. See, e.g.,Simmonds et al. 2005 Hepatology 42:962-973.

Furthermore, an “E2 polypeptide” may not be limited to a polypeptidehaving the exact sequence depicted in the HCV databases. The HCV genomeis in a state of constant flux in vivo and contains several variabledomains which exhibit relatively high degrees of variability betweenisolates. A number of conserved and variable regions are known betweenthese strains and, in general, the amino acid sequences of epitopesderived from these regions will have a high degree of sequence homology,e.g., amino acid sequence homology of more than 30%, preferably morethan 40%, more than 60%, and even more than 80-90%, or at least 95%homology or identity, when the two sequences are aligned.

Additionally, the term “E2 polypeptide” may encompass proteins, whichinclude modifications to the native sequence, such as internaldeletions, additions and substitutions (generally conservative innature), such as proteins substantially homologous to the parentsequence. These modifications may be deliberate, as throughsite-directed mutagenesis, or may be accidental, such as throughnaturally occurring mutational events. All of these modifications areencompassed in certain embodiments so long as the modified E2polypeptides function for their intended purpose. Thus, for example, ifthe E2 polypeptides are to be used in immunogenic compositions, themodifications must be such that immunological activity (i.e., theability to elicit a humoral or cellular immune response to thepolypeptide) is not lost.

“Homology” refers to the percent identity between two polynucleotide ortwo polypeptide moieties. Two DNA, or two polypeptide sequences are“substantially homologous” to each other when the sequences exhibit atleast about 50% , preferably at least about 75%, more preferably atleast about 80%-85%, preferably at least about 90%, and most preferablyat least about 95%-98% sequence identity over a defined length of themolecules. As used herein, substantially homologous also refers tosequences showing complete identity to the specified DNA or polypeptidesequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two moleculesby aligning the sequences, counting the exact number of matches betweenthe two aligned sequences, dividing by the length of the shortersequence, and multiplying the result by 100. Readily available computerprograms can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5Suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., which adapts the local homology algorithm of Smith and Waterman1981 Advances in Appl Math 2:482-489, for peptide analysis. Programs fordetermining nucleotide sequence identity are available in the WisconsinSequence Analysis Package, Version 8 (available from Genetics ComputerGroup, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs,which also rely on the Smith and Waterman algorithm. These programs arereadily utilized with the default parameters recommended by themanufacturer and described in the Wisconsin Sequence Analysis Packagereferred to above. For example, percent identity of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

Alternatively, nucleotide homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989).

Tthe term “recombinant” can be used to describe a nucleic acid moleculeand refers to a polynucleotide of genomic, RNA, DNA, cDNA, viral,semisynthetic, or synthetic origin which, by virtue of its origin ormanipulation is not associated with all or a portion of thepolynucleotide with which it is associated in nature. The term“recombinant” as used with respect to a protein or polypeptide can referto a polypeptide produced by expression of a recombinant polynucleotide.In general, the gene of interest is cloned and then expressed intransformed organisms, as described further below. The host organismexpresses the foreign gene to produce the protein under expressionconditions.

The terms “analog” and “mutein” can refer to biologically activederivatives of the reference molecule, such as E2 or an immunogenicfragment of E2, or fragments of such derivatives, that retain desiredactivity, such as immunoreactivity in assays described herein. Ingeneral, the term “analog” refers to compounds having a nativepolypeptide sequence and structure with one or more amino acidadditions, substitutions (generally conservative in nature) and/ordeletions, relative to the native molecule, so long as the modificationsdo not destroy immunogenic activity. The term “mutein” refers topolypeptides having one or more amino acid-like molecules including butnot limited to compounds comprising only amino and/or imino molecules,polypeptides containing one or more analogs of an amino acid (including,for example, unnatural amino acids, etc.), polypeptides with substitutedlinkages, as well as other modifications known in the art, bothnaturally occurring and non-naturally occurring (e.g., synthetic),cyclized, branched molecules and the like. Preferably, the analog ormutein has at least the same immunoreactivity as the native molecule.Methods for making polypeptide analogs and muteins are known in the art.

A conservative amino acid substitution in a polypeptide sequenceincludes the substitution of an amino acid in one class by an amino acidof the same class, where a class is defined by common physicochemicalamino acid side chain properties and high substitution frequencies inhomologous proteins found in nature, as determined, for example, by astandard Dayhoff frequency exchange matrix or BLOSUM matrix. Six generalclasses of amino acid side chains have been categorized and include:Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp,Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); andClass VI (Phe, Tyr, Trp). For example, substitution of an Asp foranother class III residue such as Asn, Gln, or Glu, is a conservativesubstitution. One of skill in the art can readily determine regions ofthe molecule of interest that can tolerate change by reference toHopp/Woods and Kyte—Doolittle plots. With respect to substitutions inantibodies, methods of identifying nucleotide and amino acidconservative substitutions which do not eliminate antigen binding arewell-known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187(1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burkset al. Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

An “immunogenic fragment” of a particular HCV protein will generallyinclude at least about 5-10 contiguous amino acid residues of thefull-length molecule, preferably at least about 15-25 contiguous aminoacid residues of the full-length molecule, and most preferably at leastabout 20-50 or more contiguous amino acid residues of the full-lengthmolecule, that define an epitope, or any integer between 5 amino acidsand the full-length sequence, provided that the fragment in questionretains the ability to elicit an immunological response as definedherein.

Monoclonal antibodies of the invention can be produced using a varietyof known techniques, such as the standard somatic cell hybridizationtechnique described by Kohler and Milstein (1975) Nature 256: 495, viralor oncogenic transformation of B lymphocytes or phage display techniqueusing libraries of human antibody genes. In particular embodiments, theantibodies are humanized monoclonal antibodies.

Accordingly, in one embodiment, a hybridoma method is used for producingan antibody that binds HCV E2 protein EPII (“HCV EPII”). In this method,a mouse or other appropriate host animal can be immunized with asuitable antigen in order to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theantigen used for immunization. In some embodiments, the antigen isPeptide A, Peptide B, or Peptide B-short. Alternatively, lymphocytes maybe immunized in vitro. Lymphocytes can then be fused with myeloma cellsusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,pp. 59-103 (Academic Press, 1986)). Culture medium in which hybridomacells are growing is assayed for production of monoclonal antibodiesdirected against the antigen. After hybridoma cells are identified thatproduce antibodies of the desired specificity, affinity, and/oractivity, the clones may be subcloned by limiting dilution proceduresand grown by standard methods (Goding, Monoclonal Antibodies:Principlesand Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture mediafor this purpose include, for example, D-MEM or RPMI-1640 medium. Inaddition, the hybridoma cells may be grown in vivo as ascites tumors inan animal. The monoclonal antibodies secreted by the subclones can beseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

As discussed further below, a hybridoma cell producing monoclonalantibody #41 and a hybridoma cell producing monoclonal antibody #8 aredisclosed herein.

The binding specificity to HCV E2 protein EPII of monoclonal antibodies,or fragments thereof, prepared using any technique including thosedisclosed herein, can be determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). The binding affinity of a monoclonalantibody or portion thereof also can be determined by the Scatchardanalysis of Munson et al., Anal. Biochem., 107:220 (1980).

In certain embodiments, an antibody binding HCV E2 protein EPII may befurther altered or optimized to achieve a desired binding specificityand/or affinity using art recognized techniques, such as those describedherein.

In one embodiment, partial antibody sequences derived from a givenantibody may be used to produce structurally and functionally relatedantibodies. For example, antibodies interact with target antigenspredominantly through amino acid residues that are located in the sixheavy and light chain complementarity determining regions (CDRs). Forthis reason, the amino acid sequences within CDRs are more diversebetween individual antibodies than sequences outside of CDRs. BecauseCDR sequences are responsible for most antibody-antigen interactions, itis possible to express recombinant antibodies that mimic the propertiesof specific naturally occurring antibodies by constructing expressionvectors that include CDR sequences from the specific naturally occurringantibody grafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998, Nature332:323-327; Jones, P. et al., 1986, Nature 321:522-525; and Queen, C.et al., 1989, Proc. Natl. Acad. See. U.S.A. 86:10029-10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline antibody gene sequences.

Thus, one or more structural features of an anti-HCV EPII antibodydisclosed herein, such as the CDRs, can be used to create structurallyrelated anti-HCV EPII antibodies that retain at least one functionalproperty of the antibodies of the invention, e.g., inhibiting infectionof cells exposed to HCV.

Antibody heavy and light chain CDR3 domains are known to play aparticularly important role in the binding specificity/affinity of anantibody for an antigen. Accordingly, in certain embodiments, antibodiesare generated that include the heavy and/or light chain CDR3s of theparticular antibodies described herein. The antibodies can furtherinclude the heavy and/or light chain CDR1 and/or CDR2s of the antibodiesdisclosed herein.

The CDR 1, 2, and/or 3 regions of the engineered antibodies describedabove can comprise the exact amino acid sequence(s) as those disclosedherein. However, the ordinarily skilled artisan will appreciate thatsome deviation from the exact CDR sequences may be possible,particularly for CDR1 and CDR2 sequences, which can tolerate morevariation than CDR3 sequences without altering epitope specificity (suchdeviations are, e.g., conservative amino acid substitutions).Accordingly, in another embodiment, the engineered antibody may becomposed of one or more CDR1s and CDR2s that are, for example, 90%, 95%,98%, 99% or 99.5% identical to the corresponding CDRs of an antibodynamed herein.

In another embodiment, one or more residues of a CDR may be altered tomodify binding to achieve a more favored on-rate of binding. Using thisstrategy, an antibody having ultra high binding affinity of, forexample, 10¹⁰ M⁻¹ or more, can be achieved. Affinity maturationtechniques, well known in the art and those described herein, can beused to alter the CDR region(s) followed by screening of the resultantbinding molecules for the desired change in binding. Accordingly, asCDR(s) are altered, changes in binding affinity as well asimmunogenicity can be monitored and scored such that an antibodyoptimized for the best combined binding and low immunogenicity areachieved.

Modifications can also be made within one or more of the framework orjoining regions of the heavy and/or the light chain variable regions ofan antibody, so long as antigen binding affinity subsequent to thesemodifications is better than 10⁶ M⁻¹.

In another embodiment, the antibody is further modified with respect toeffector function, so as to enhance the effectiveness of the antibody intreating cancer, for example. For example cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region.

In another aspect, a composition, e.g., a pharmaceutical composition, isdisclosed herein. The composition can contain one or a combination ofmonoclonal antibodies, (or antigen-binding fragments thereof),formulated together with a pharmaceutically acceptable carrier. In oneembodiment, the composition includes an isolated antibody that binds HCVEPII. In an embodiment, the composition contains an isolated antibody orfragment thereof disclosed herein and at least one additionaltherapeutic agent. The therapeutic agent can be a small molecule drug,or a biological such as a hormone, a protein, or another antibody orfragment thereof. In some embodiments, the composition further comprisesa pharmaceutically acceptable carrier. A “therapeutic agent” means asubstance that when administered to a patient provides any therapeuticbenefit. A therapeutic benefit may be an amelioration of symptoms of HCVinfection or prevention of HCV infection.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody, may becoated in a material to protect the compound from the action of acidsand other natural conditions that may inactivate the compound.

Compositions can be administered alone or in combination therapy, i.e.,combined with other agents. For example, the combination therapy caninclude a composition provided herein with at least one or moreadditional therapeutic agents, such as an anti-viral agent describedherein, or another antibody.

Compositions can be administered by a variety of methods known in theart. As will be appreciated by the skilled artisan, the route and/ormode of administration will vary depending upon the desired results. Theantibodies can be prepared with carriers that will protect theantibodies against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J.R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

To administer compositions by certain routes of administration, it maybe necessary to coat the constituents, e.g., antibodies, with, orco-administer the compositions with, a material to prevent itsinactivation. For example, the compositions may be administered to asubject in an appropriate carrier, for example, liposomes, or a diluent.Acceptable diluents include saline and aqueous buffer solutions.Liposomes include water-in-oil-in-water CGF emulsions as well asconventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Acceptable carriers include sterile aqueous solutions or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. The use of such media and agents forpharmaceutically active substances is known in the art. Except insofaras any conventional media or agent is incompatible with the antibodies,use thereof in compositions provided herein is contemplated.Supplementary active constituents can also be incorporated into thecompositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Including inthe composition an agent that delays absorption, for example,monostearate salts and gelatin can bring about prolonged absorption ofthe injectable compositions.

Sterile injectable solutions can be prepared by incorporating themonoclonal antibodies in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by sterilization microfiltration. Generally, dispersions areprepared by incorporating the antibodies into a sterile vehicle thatcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. For example, human antibodiesmay be administered once or twice weekly by subcutaneous injection oronce or twice monthly by subcutaneous injection.

It can be advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the subjects to be treated; each unit contains apredetermined quantity of antibodies calculated to produce the desiredtherapeutic effect in association with the required pharmaceuticalcarrier. The specification for the dosage unit forms provided herein aredictated by and directly dependent on (a) the unique characteristics ofthe antibodies and the particular therapeutic effect to be achieved, and(b) the limitations inherent in the art of compounding such antibodiesfor the treatment of sensitivity in individuals.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations include those suitablefor oral, nasal, topical (including buccal and sublingual), rectal, andparenteral administration. Parenteral administration is the most commonroute of administration for therapeutic compositions comprisingantibodies. The formulations may conveniently be presented in unitdosage form and may be prepared by any methods known in the art ofpharmacy. The amount of antibodies that can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Thisamount of antibodies will generally be an amount sufficient to produce atherapeutic effect. Generally, out of one hundred per cent, this amountwill range from about 0.001 per cent to about ninety percent of antibodyby mass, preferably from about 0.005 per cent to about 70 per cent, mostpreferably from about 0.01 per cent to about 30 per cent.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions provided herein includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Particularexamples of adjuvants which are well-known in the art include, forexample, inorganic adjuvants (such as aluminum salts, e.g., aluminumphosphate and aluminumhydroxide), organic adjuvants (e.g., squalene),oil-based adjuvants, virosomes (e.g., virosomes which contain amembrane-bound heagglutinin and neuraminidase derived from the influenzavirus).

Prevention of presence of microorganisms may be ensured both bysterilization procedures, supra, and by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

When compositions are administered as pharmaceuticals, to humans andanimals, they can be given alone or as a pharmaceutical compositioncontaining, for example, 0.001 to 90% (more preferably, 0.005 to 70%,such as 0.01 to 30%) of active ingredient in combination with apharmaceutically acceptable carrier.

Regardless of the route of administration selected, compositionsprovided herein, may be used in a suitable hydrated form, and they maybe formulated into pharmaceutically acceptable dosage forms byconventional methods known to those of skill in the art.

Actual dosage levels of the antibodies in the pharmaceuticalcompositions provided herein may be varied so as to obtain an amount ofthe active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions employed, or the ester, saltor amide thereof, the route of administration, the time ofadministration, the rate of excretion of the particular compound beingemployed, the duration of the treatment, other drugs, compounds and/ormaterials used in combination with the particular compositions employed,the age, sex, weight, condition, general health and prior medicalhistory of the patient being treated, and like factors well known in themedical arts. A physician or veterinarian having ordinary skill in theart can readily determine and prescribe the effective amount of thecomposition required. For example, the physician or veterinarian couldstart doses of the antibodies at levels lower than that required toachieve the desired therapeutic effect and gradually increasing thedosage until the desired effect is achieved. In general, a suitabledaily dose of compositions provided herein will be that amount of theantibodies which is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. It is preferred that administration be intravenous,intramuscular, intraperitoneal, or subcutaneous, preferably administeredproximal to the site of the target. If desired, the effective daily doseof a therapeutic composition may be administered as two, three, four,five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms. While itis possible for antibodies to be administered alone, it is preferable toadminister antibodies as a formulation (composition).

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition can be administered with a needleless hypodermic injectiondevice, such as the devices disclosed in U.S. Pat. Nos. 5,399,163,5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.Examples of well-known implants and modules useful in methods disclosedherein include: U.S. Pat. No. 4,487,603, which discloses an implantablemicro-infusion pump for dispensing medication at a controlled rate; U.S.Pat. No. 4.,486,194, which discloses a therapeutic device foradministering medications through the skin; U.S. Pat. No. 4,447,233,which discloses a medication infusion pump for delivering medication ata precise infusion rate; U.S. Pat. No. 4,447,224, which discloses avariable flow implantable infusion apparatus for continuous drugdelivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Many othersuch implants, delivery systems, and modules are known to those skilledin the art.

In certain embodiments, the monoclonal antibodies can be formulated toensure proper distribution in vivo. For example, the therapeutic can beformulated in liposomes. Methods of manufacturing liposomes are known inthe art. The liposomes may comprise one or more moieties that areselectively transported into specific cells or organs, thus enhancetargeted drug delivery.

Also provided are methods of using antibodies (and antigen bindingfragments thereof) that bind HCV EPII in a variety of ex vivo and invivo diagnostic and therapeutic applications involving HCV.

Accordingly, in one embodiment, the antibody or a fragment thereofspecifically binding HCV EPII can be used to detect HCV in a sample. Insome embodiments, the antibody or a fragment thereof specificallybinding HCV EPII can be used to detect HCV genotype 1a in a sample. Inan embodiment, the method comprises contacting the antibody or fragmentthereof with a sample under conditions such that the antibody binds HCVEPII; and detecting antibody bound to HCV EPII. Such a method could be acomponent of a diagnostic method for HCV infection or for a method ofidentifying the genotype of HCV infection, for example to optimizetreatment. In one embodiment, a method is provided for treating orpreventing HCV infection by administering to a subject an HCVneutralizing antibody disclosed herein. The HCV neutralizing antibodycan be administered alone or in combination with one or more additionaltherapeutic agents. The HCV neutralizing antibody can be administered inan amount effective to treat or prevent HCV infection. In someembodiments, the subject can be a liver transplant patient, specificallythe liver transplant patient can have chronic hepatitis C. A “livertransplant patient” is a patient in any stage associated with obtaininga liver transplant, including for example a patient with liver diseaseevaluated as needing a liver transplant, a patient scheduled for a livertransplant, or a patient post-liver transplant.

The term “effective amount,” as used herein, refers to that amount of anantibody or an antigen binding fragment thereof that binds HCV E2protein EPII, which is sufficient to effect treatment or prevent HCVinfection, as described herein, when administered to a subject.Therapeutically effective amounts of antibodies of the present inventionwill vary depending upon the relative activity of the antibodies (e.g.,in inhibiting HCV infection of cells) and depending upon the subject anddisease condition being treated, the weight and age of the subject, theseverity of the disease condition, the manner of administration and thelike, which can readily be determined by one of ordinary skill in theart. The dosages for administration can range from, for example, about 1ng to about 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng toabout 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg,about 100 ng to about 6,000 mg, about 200 ng to about 5,500 mg, about300 ng to about 5,000 mg, about 400 ng to about 4,500 mg, about 500 ngto about 4,000 mg, about 1 μg to about 3,500 mg, about 5 μg to about3,000 mg, about 10 _(l)ig to about 2,600 mg, about 20 _(l)ig to about2,575 mg, about 30 μg to about 2,550 mg, about 40 _(l)ig to about 2,500mg, about 50 _(l)ig to about 2,475 mg, about 100 μpg to about 2,450 mg,about 200 μg to about 2,425 mg, about 300 μg to about 2,000, about 400μg to about 1,175 mg, about 500 μg to about 1,150 mg, about 0.5 mg toabout 1,125 mg, about 1 mg to about 1,100 mg, about 1.25 mg to about1,075 mg, about 1.5 mg to about 1,050 mg, about 2.0 mg to about 1,025mg, about 2.5 mg to about 1,000 mg, about 3.0 mg to about 975 mg, about3.5 mg to about 950 mg, about 4.0 mg to about 925 mg, about 4.5 mg toabout 900 mg, about 5 mg to about 875 mg, about 10 mg to about 850 mg,about 20 mg to about 825 mg, about 30 mg to about 800 mg, about 40 mg toabout 775 mg, about 50 mg to about 750 mg, about 100 mg to about 725 mg,about 200 mg to about 700 mg, about 300 mg to about 675 mg, about 400 mgto about 650 mg, about 500 mg, or about 525 mg to about 625 mg, of anantibody or antigen binding portion thereof, according to the invention.Dosage regimens may be adjusted to provide the optimum therapeuticresponse. An effective amount is also one in which any toxic ordetrimental effects (i.e., side effects) of an antibody or antigenbinding fragment thereof are minimized and/or outweighed by thebeneficial effects.

The antibody can be administered alone or with another therapeutic agentthat acts in conjunction with or synergistically with the antibody totreat or prevent HCV infection. Such therapeutic agents include thosedescribed herein, for example, small organic molecules, monoclonalantibodies, and recombinantly engineered biologics.

Also provided are kits comprising one or more anti-HCV EPII antibodies(or antigen binding fragments thereof), optionally contained in a singlevial, and include, e.g., instructions for use in treating or preventingHCV infection. The kits may include a label indicating the intended useof the contents of the kit. The term label includes any writing,marketing materials or recorded material supplied on or with the kit, orwhich otherwise accompanies the kit.

Other embodiments of the present invention are described in thefollowing non-limiting Examples.

EXAMPLES

Materials and Methods

Peptide synthesis. All peptides were chemically synthesized by the CoreLaboratory of the Center for Biologics Evaluation and Research at the USFood and Drug Administration, with an Applied Biosystems (Foster City,CA) Model 433A peptide synthesizer. Biotinylated peptides weresynthesized with Fmoc-Lys (Biotin-LC)-Wang resin (AnaSpec, San Jose, CA)as described previously (Zhang P et al. 2007).

ELISA. Biotin-conjugated peptide (200 ng/well) was added tostreptavidin-coated 96-well Maxisorp plates (Pierce) and incubated atroom temperature for 1 hour (h) in Super Block Blocking Buffer (ThermoScientific). The wells were blocked further in blocking buffer foranother hour at 37° C. After washing the plate 4× with phosphatebuffered saline (PBS) buffer pH 7.4 containing 0.05% Tween-20 to removeunbound peptides, serial dilutions of the test antibodies were added tothe plate and incubated at 37° C. for 1 h. The plate was then washed 4×before the secondary monoclonal antibody, either a goat anti-mouseperoxidase-conjugated IgG or a goat anti-human peroxidase-conjugated IgG(Sigma-Aldrich) at a 1:5000 dilution, was added to the wells andincubated at 37° C. for lh. After 4 washes, the reaction was developedwith ABTS peroxidase substrate (KPL, Gaithersburg, MD) and stopped byadding 100 μL of a 1% SDS solution, or the reaction was developed with1-Step TMB-ELISA substrate solution (KPL, Gaithersburg, MD) and stoppedby adding 100 μL 4N Sulfuric Acid. The absorbance of each well wasmeasured at 405 nm and 450 nm, respectively, using a SpectraMax M2emicroplate reader (Molecular Devices).

Neutralization assay. Virus stocks were prepared by transfectingfull-length HCV RNA derived from an HCV genotype 2a clone, J6/JFH1 (agift from Charles Rice, Rockefeller University), into Huh 7.5 cells aspreviously described (Duan H et al. 2010. Vaccine. 28:4138-4144, Zhanget al. 2007, Zhang P et al. 2009). An HCV genotype 1a/2a chimera viruswas produced by replacing the structural genes of J6/JFH1 with that ofthe HCV H strain (H77), which is known to be genotype 1a. Briefly, Huh7.5 cells were seeded at a density of 4-5×103 cells/well in 96-wellplates to obtain approximately 60% confluence in 24 h. The virus stockwas diluted in DMEM supplemented with 10% fetal bovine serum (FBS)/1%penicillin/streptomycin/2 mM glutamine to yield approximately 50infected foci per well in the absence of antibodies. Viruses were mixedwith a diluted antibody or with cell culture medium, incubated at 37° C.for 1 h, and then inoculated into Huh 7.5 cells. After 3 days inculture, virus foci were detected either by immunofluorescence orimmunoperoxidase staining and then counted. Neutralization wasdetermined by comparing the infectivity of the viruses incubated withthe antibody to the infectivity of the viruses incubated with mediumalone or with pre-immune plasma. The median 50% inhibitory dilution(ID5o) was determined according to the method of Reed and Muench (1938.Am. J. Hyg. 27:493-497). Statistical analysis was performed withGraphPad Prism 4 (GraphPad Software, La Jolla, CA) by using the unpairedt-test with two-tailed P value (P value <0.05). Error bars represent thestandard deviation or the standard error of the mean.

Enrichment and removal of peptide-specific antibodies. 500 ng ofbiotinylated Peptide B, Peptide D or an unrelated peptide control (apool of overlapping peptides representing the M2 protein from theInfluenza virus) was mixed with 100 μL of streptavidin-coated Dynabeads(Invitrogen, Grand Island, N.Y.) and incubated at room temperature for 1h. After washing with PBS (pH 7.4), the beads were mixed with anappropriate dilution of ascites fluid or plasma, which containedspecific antibodies, and incubated at room temperature for 1 h. Toenrich for peptide-specific antibodies, the beads were collected with amagnet stand. After washing the beads with PBS, the antibodies wereeluted from the beads with Glycine-HCl solution (pH 2.2). The eluateswere neutralized by mixing with an equal volume of Tris-HCl buffer (pH9.2). In contrast, to remove the peptide-specific antibodies, the beadswere pelleted with a magnet stand and the supernatant was collected forfurther analysis.

Phage display. The selection of peptides from random peptide phagedisplay libraries (New England Biolabs, Beverly, Mass.) was describedpreviously (Zhang P et al. 2007). Briefly, 10¹⁰ phages were incubatedwith individual monoclonal antibody/protein G mixtures at roomtemperature for 20 min. After 8 washes with 0.05 M Tris-HCl buffer (pH7.5) containing 0.15 M NaCl and 0.05% Tween-20, the phages were elutedfrom the complexes with 0.1 M HCl and neutralized with 1 M Tris-HClbuffer (pH 9.0). The eluted phages were then amplified in the hoststrain ER2738 for 4-5 h. After three additional rounds of selection ofamplified phages by the same monoclonal antibody, the DNA from eachsingle-phage plaque was sequenced, and the corresponding peptidesequence was then deduced from the DNA sequence.

Statistical analysis: Statistical analysis was performed with GraphPadPrism 4 using unpaired t-test with two-tailed P value (P value<0.05).Error bars represent the standard deviation or the standard error of themean.

Example 1 Generation of Monoclonal Antibodies

Monoclonal antibodies were produced using the standard procedures ofHarlan Bioproducts for Science (Indianapolis, Ind.). Briefly, Balb/cmice were injected intraperitoneally (i.p.) with a chemicallysynthesized Peptide A (amino acid residues 412-447 of the E2 proteinfrom the HCV H strain (H77) (FIG. 1A), which was conjugated to keyholelimpet hemocyanin (KLH). Mice that produced high titers of antibody toPeptide A were selected for cell fusion to generate hybridomas.Antibody-positive cells were cloned by the limiting dilution method forseveral cycles. At each cloning cycle, the tissue culture supernatant ofeach clone was screened by ELISA for the presence of antibodies toPeptide B (Epitope II). The selected anti-Peptide B-positive clones wereinjected i.p. into Balb/c mice primed with pristane (Sigma-Aldrich) toproduce ascites fluid.

A panel of hybridoma cell lines that produced monoclonal antibodies wasobtained after immunizing the mice with Peptide A (a.a. 412-447) (FIG.1A). Using an ELISA, seven monoclonal antibodies (ascites fluid) fromthe panel that bound to Peptide A were identified (FIG. 1B).Biotin-conjugated Peptide A (200 ng/well) was added tostreptavidin-coated 96-well plates. Each monoclonal antibody (ascitesfluid) was diluted 1:1000 and used as the primary antibody. The resultsare shown in FIG. 1B in which the y axis indicates absorbance at 405 nmobtained in the ELISA, representing specific binding of a given antibodyto Peptide A. Data shown represent the mean of three independentexperiments, with the standard deviation indicated by the error barextending from the top of each bar.

A similar ELISA was performed to detect specific binding of the panel ofantibodies to Peptide B. Biotin-conjugated Peptide B (200 ng/well) wasadded to the streptavidin-coated 96-well plates. The ELISA conditionswere similar to those in the Peptide A-specificity experiment. The data,shown in FIG. 1C, also represent at least 3 independent experiments.

The binding capacity toward Peptide A of the seven antibodies wascomparable at a dilution of 1:1000 (FIG. 1B). Four of the sevenantibodies, namely, #8, #12, #41 and #50, were also shown to bindspecifically to Peptide B (FIG. 1A and C), while the other threeantibodies, #27, #48 and #49 did not bind to Peptide B (FIG. 1C).

Example 2 Neutralization and Non-Neutralization of HCV 1a/2a Chimeras byMonoclonal Antibodies.

The monoclonal antibodies that were capable of binding to the Peptide Bregion of the E2 protein were tested for their capacity to neutralizeHCV infection in a cell culture system. A genotype 1a/2a chimeric HCVwas used for the neutralization assay because the peptide sequence ofthe genotype 1a virus was used to design the immunogen for generatingthese monoclonal antibodies.

Each antibody (ascites fluid, 1:200) was incubated with appropriatelydiluted genotype 1a/2a virus, before adding the mixture to Huh 7.5cells. Cell culture medium (DMEM) was used as a negative control for theantibody. The results are shown in FIG. 2A in which the x axis indicatesthe particular antibody tested in the experiment and the y axisindicates the relative infectivity of the virus (%), i.e., percent ofthe negative control. Each bar represents the mean of at least 3independent experiments with the error bar showing the standard error ofthe mean indicated at the top of each bar.

As shown in FIG. 2A, antibodies #8 and #41 were able to neutralize thegenotype 1a/2a virus. In contrast, antibodies #12 and #50, which alsorecognized Peptide B in an ELISA (FIG. 1C), were unable to neutralizethe virus.

To ascertain whether the observed neutralization of the 1a/2a chimericvirus was Peptide B-specific, adsorption experiments were performed todeplete Peptide B-specific binding activity from the ascites fluid thatcontained neutralizing antibody #41. Antibody #41 was adsorbed with (+)or without (-) Peptide B prior to performing an ELISA to test itsbinding to Peptide B (left panel of FIG. 2B), and a neutralization assayto assess its neutralizing activity in Huh 7.5 cells (right panel ofFIG. 2B). Each of the samples shown on the x axis was tested at adilution of 1:10⁵ in an ELISA. The y axis indicates the absorbance at405 nm obtained in an ELISA, representing the specific binding of agiven antibody to Peptide B. The data shown represent at least 3independent experiments. The standard deviation of the assay isindicated. For the neutralization assay (right panel), the supernatantwas diluted at 1:400, and incubated with the genotype 1a/2a virus beforeadding the mixture to Huh 7.5 cells. The cell culture medium (Med) wasused as the negative control against the tested antibodies. The x axisindicates the samples tested in this assay. The y axis indicates therelative infectivity of the virus (%), i.e., percent of the negativecontrol. The statistical significance of the difference in infectivityis also indicated.

As demonstrated by the ELISA results shown in FIG. 2B (left panel), thePeptide B-specific binding activity could be substantially absorbed outby Peptide B. Concurrently, its neutralizing activity was alsosignificantly diminished (FIG. 2B, right panel).

The two Peptide B-binding antibodies showing neutralization of genotype1a/2a were tested for their ability to neutralize other genotypes.Serially diluted antibodies were tested against J6/JFH1, a genotype 2avirus, 1a/2a, 1b/2a and 3a/2a genotypes in Huh 7.5 cells with the sameprocedure described above. The results are shown in FIG. 2C.Neutralizing antibodies #8 and #41 were not able to neutralize thegenotype 2a virus, J6/JFH1 (FIG. 2C), or other chimeric viruses 1b/2aand 3a/2a. These results demonstrated that antibodies #8 and #41,through the direct binding of Peptide B, could only neutralize HCV in agenotype 1a virus-specific manner.

Example 3 Neutralization of HCV by Antibody #41 in the Presence orAbsence of Non-Neutralizing Antibodies

The ability of antibody #41 to neutralize genotype 1a/2a virus wasmeasured in the presence or absence of non-neutralizing antibody #12 or#50. Neutralizing antibody #41 was diluted at 1:400 in DMEM, and thenmixed with antibody #12 or #50 at ratios of 1:1 and 1:4 (v/v). Theantibody mixture was subsequently incubated with genotype 1a/2a chimericvirus at 37° C. for 1 h before being added to Huh 7.5 cells. The resultsfrom 3 independent experiments are presented in FIG. 3. Neither antibody#12 nor #50 showed any interfering effect on the neutralizing ability ofmonoclonal antibody #41 (FIG. 3). Similar results were obtained inexperiments with antibody #8.

Example 4 Residue-Specific Binding of Neutralizing and Non-NeutralizingAntibodies

The residues involved in the binding of the four Peptide B-bindingantibodies were mapped by screening random peptide phage displaylibraries. The amino acid sequences of phage clusters identified afterat least 3 rounds of screening phage-display libraries (12-mer and7-mer) with neutralizing antibodies #8 and #41 are shown in FIG. 4 alongwith the number of specific peptides sequenced/the total number ofpeptides sequenced (shown in parenthesis).

To further determine the residue-specificity of these antibodies, eachof the binding residues identified by the phage display analysis weresubstituted one at a time by an Alanine residue in a truncated versionof Peptide B (“ B short” ; a.a. 434-446 of SEQ ID NO:1) (see FIG. 5),and then tested by ELISA to determine the effect of the specificsubstitution on the binding of these antibodies. Biotin-conjugatedpeptides were chemically synthesized to represent B short and itsmutations. The B short mutant peptides shown in FIG. 5 contained asingle alanine (A) substitution at positions 437, 438, 440, 441 and 442,respectively.

In the ELISA, biotin-conjugated B short peptide and its mutants wereadded to streptavidin-coated 96-well plates at 200 ng/well. Themonoclonal antibody (ascites fluid) was diluted at 1:10⁵ dilution, andapplied as the primary antibody. Phosphate buffered saline (PBS) wasincluded as the negative control. The results of the ELISA usingantibody #41 are shown in FIG. 5. Similar experiments were performedwith neutralizing antibody #8 and nonneutralizing antibodies #12 and#50.

Residues determined to be involved in binding for each of the fourantibodies are summarized in FIG. 6A.

All four antibodies tested in this study, irrespective of theirneutralizing function, lost their binding to B short when W437 or L438was replaced by an alanine residue, thus confirming that positions 437and 438 were core contact residues recognized by these antibodies.Neutralizing antibodies #8 and #41 were less affected by thesubstitution at positions 441 and 442 in contrast to the effect on thebinding capacities of the non-neutralizing antibodies #12 and #50.

HCV genotype 1a has a W437 in its E2 protein, whereas the other HCVgenotypes often contain an F residue at the same position (FIG. 6B). Wehypothesized that the genotype 1a-specific neutralizing antibodies arelimited to one genotype because of their inability to recognize F437. Totest this hypothesis, we substituted the W residue at position 437 inthe truncated Peptide B (B short) with an F residue and then evaluatedits effect on binding in an ELISA experiment. The biotin-conjugatedpeptides chemically synthesized for this experiment to represent PeptideB (residues 427-446 of SEQ ID NO:1), the truncated Peptide B (B short)(residues 434-446 of SEQ ID NO:1), and B short sequences with theindicated specific single mutations at position 437 are shown in FIG.7A. A hyphen indicates an amino acid residue identical to that of theH77 sequence.

Biotin-conjugated peptide B, B short peptide, or a B short mutans wasadded to streptavidin-coated 96-well plates at 200 ng/well. Eachmonoclonal antibody (ascites fluid) was used at a 1:10⁵ dilution as theprimary antibody in the ELISA. Cell culture medium was used as thenegative control of the antibody. The ELISA results are shown in FIG.7B.

The W437F switch resulted in a loss of binding by neutralizingantibodies #41 and #8 to B short (FIG. 7B), as well as innon-neutralizing antibodies #12 and #50. This result confirmed that thepresence of W437 was required for the shared recognition by theseantibodies, and further indicated that W437 might be indispensable forthe observed genotype 1a-specific neutralization of HCV shown byantibodies #41 and #8.

Example 5 Sequencing of Mouse Monoclonal Antibody (mAb) #41

About 5×10⁷ of #41 hybridoma cells were used to extract the mRNA withTrizol and chloroform. Reverse transcriptase (RT) reaction was performedwith SUPERSCRIPT® III First-Strand Synthesis Supermix Kit (LifeTechnologies) and multiple primers for the kappa and heavy chains,respectively. PCR was performed using multiple sets of primers for thekappa and heavy chains. The PCR amplification products were confirmedand purified using 1% agarose and then sequenced. Sequence results frommultiple primers were aligned and analyzed using IMGT/V-QUEST (Brochet,X., et al. Nucl. Acids Res, 36, W503-508 (2008)), available on theinternet from IIMGT®, the International ImMunoGeneTics InformationSystem® Lefranc, M.-P., et al. Nucl. Acids Res, 37, D1006-D1012 (2009)).Nucleotide sequences of the heavy chain and the light (kappa) chain areSEQ ID NOS: 4 and 5, respectively. Translation of SEQ ID NOS: 4 and 5yield protein sequences of the heavy chain and the light (kappa) chainof SEQ ID NOS: 2 and 3, respectively. FIG. 8 shows the nucleotidesequence and translated protein sequence of the kappa chain and theheavy chain of antibody #41.

The terms “a” and “an” do not denote a limitation of quantity, butrather denote the presence of at least one of the referenced item. Theterm “or” means “and/or” . The terms “comprising” , “having” ,“including” , and “containing” are to be construed as open-ended terms(i.e., meaning “including, but not limited to”). The modifier “about”used in connection with a quantity is inclusive of the stated value andhas the meaning dictated by the context (e.g., includes the degree oferror associated with measurement of the particular quantity).

Recitation of ranges of values are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. The endpoints of all ranges are includedwithin the range and independently combinable.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof these embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

The invention claimed is:
 1. An isolated antibody or fragment thereofspecifically binding to hepatitis C virus (HCV) E2 protein Epitope II,the antibody or fragment thereof comprising a heavy chain variableregion comprising complementarity determining region (CDR) amino acidsequences CDR1 comprising residues 25-32 (GYSFTNYY) of SEQ ID NO:2, CDR2comprising residues 50-57 (IFPGGGNT) of SEQ ID NO:2, and CDR3 comprisingresidues 96-107 (SRDIY GDAWFAY) of SEQ ID NO:2; and a light chainvariable region comprising CDR amino acid sequences CDR1 comprisingresidues 27-37 (Q NIVHRNGNTY) of SEQ ID NO:3, CDR2 comprising residues55-57 (KVS) of SEQ ID NO:3, and CDR3 comprising residues 94-102 (FQGSHFPPT) of SEQ ID NO:3.
 2. The antibody or fragment thereof of claim 1,wherein the heavy chain variable region comprises the amino acidsequence of SEQ ID NO: 2, and the light chain variable region comprisesthe amino acid sequence of SEQ ID NO:
 3. 3. The antibody or fragmentthereof of claim 1 binding specifically to at least residues 434-446 ofHCV E2 protein Epitope II (EP II), EPII comprising residues 427-446 ofSEQ ID NO:1.
 4. The isolated antibody or fragment thereof of claim 1,wherein the antibody is a monoclonal antibody.
 5. The isolated antibodyor fragment thereof of claim 1, wherein the antibody is a humanizedantibody.
 6. The isolated antibody or fragment thereof of claim 1,wherein the HCV E2 protein Epitope II (EP II) comprises W⁴³⁷.
 7. Theisolated antibody or fragment thereof of claim 2, wherein the heavychain variable region is encoded by SEQ ID NO:4.
 8. The isolatedantibody or fragment thereof of claim 2, wherein the light chainvariable region is encoded by SEQ ID NO:5.
 9. The antibody or fragmentthereof of claim 1, wherein the antibody or fragment thereof neutralizesHCV genotype 1 a in a cell culture system.
 10. A composition comprisingthe antibody or fragment thereof of claim 1; and a pharmaceuticallyacceptable carrier.
 11. A composition comprising the antibody orfragment thereof of claim 1, wherein the antibody or fragment thereof islinked to a toxic material, a chemotherapeutic agent, or a labelingmaterial.
 12. A method of detecting hepatitis C virus (HCV) E2 proteinEpitope II in a sample comprising contacting the antibody of claim 1with a sample under conditions such that the antibody binds an HCV E2protein Epitope II (EP II) sequence comprising at least residues 427-446of SEQ ID NO:1; and detecting antibody bound to EP II.
 13. A method oftreating or preventing HCV infection comprising administering theantibody or fragment thereof according to claim 1 to a subject exposedto or infected with HCV.
 14. The method of claim 13, wherein the subjectis a liver transplant patient.